This application is for continuation of our researh program into the mechanism of mineralization in bone and dentin. These studies are important for understanding, in humans, bone and tooth growth, development, tissue remodeling, and pathologies such as dentinogenesis and osteogenesis imperfecta, ectopic calcifications and other skeletal problems as well as for the design of engineered biomimetic bone and tooth replacements and devices with appropriate tissue strength and toughness. Invertebrate mineralization shares similar mechanisms. Thus, comparing verebrate and invertebrate systems may provide a deeper understanding of the relationships between matrix structure and the proteins guiding mineralization. In dentin and bone unique proteins have been shown to control the placement, nucleation, orientation and growth of the mineral crystals. Similar matrix proteins, some related by strong sequence similarities, carry out these functions in invertebrate tissues. The primary aims of the present grant focused specifically on contrasting the structures and composition of the mineral and organic phases of intact bovine peritubular dentin (PTD) and intertubular dentin (ITD) using advanced techniques such as TOF-SIMS, synchrotron micro CT scanning and various forms of TEM-SEM. Similarly, urchin teeth were found to contain two distinct mineral phases of differing protein and mineral compositions. Both sets of data indicated the need to proceed to higher resolution with new technology in separating PTD from ITD and separating the urchin high and low Mg calcites, The new specific aims are: 1A) To isolate the intact PTD and ITD and compare the structures and compositions of their mineral and organic phases at the micro to nano level using new laser capture- dissection and milling techniques, coupled with biochemical studies of the isolated PTD proteophospholipid. 1B) To compare the structure/compositions of coronal and radicular PTD. 2A). To identify the mineral-related proteins (MRP)of the urchin tooth. 2B) Prepare antibodies and localize the MRP within the tooth. 2C ) To correlate the tooth mineral element structures with their protein and mineral ion content, and 2D) use laser microdissection to separate high Mg calcite primary plates from the very high Mg cementing columns and determine the role of the proteins in regulating mineral ion deposition.